The invention relates to nucleic acids which encode plant polypeptides with the biological activity of phosphomevalonate kinases, to the polypeptides encoded by them and their use as targets for herbicides and their use for identifying novel, herbicidally active compounds, and to methods for finding modulators of these polypeptides.
Unwanted plant growth can be prevented by using herbicides. The demands made on herbicides with regard to their efficacy, costs and environmental compatibility have been steadily increasing. There exists therefore a demand for new substances which can be developed into potent new herbicides. In general, it is usual to search for such new lead structures in greenhouse tests. However, such tests are laborious and expensive. Accordingly, the number of substances which can be tested in the greenhouse is limited.
Advantageous targets for herbicides are searched for in essential biosynthetic pathways. Thus, the biosynthesis of isoprenoids in plants leads, inter alia, to the synthesis of carotenoids and of the side chains of plastoquinone and of chlorophyll. These products are essential for the photosynthetic growth of plants. The inhibition of one step in this biosynthetic pathway leads to the termination of a plant""s growth. Moreover, plant hormones such as gibberellic acid, abscisic acid and brassinosteroids and membrane components (phytosterols), which are also essential for the plant""s growth, are formed from isoprenoids.
Isopentyl diphosphate (IPP) is the branching point from which the widest range of isoprenoids are formed. The production of IPP is therefore a critical point in plant metabolism. In plants, IPP is produced via two different metabolic pathways in different compartments. In the endoplasmic reticulum (ER) and in the cytosol, IPP synthesis proceeds via the classic acetate/mevalonate metabolic pathway as it also proceeds in the animal organism. In contrast, IPP is synthesized in chloroplasts via the alternative glyceraldehyde phosphate/pyruvate metabolic pathway. Both metabolic pathways are essential since various isoprenoid metabolites are formed in the different compartments. Moreover, the degree to which the two metabolic pathways are autonomous or to which an exchange of metabolites takes place between the compartments has not been elucidated as yet (Heintze et al., 1990, Kleinig, 1989). (See References section below for full citation to these and other references referred to herein).
Clomazone is a known herbicidal compound which reduces the carotenoid and chlorophyll content in the leaf. For a long time it has been assumed that clomazone acts via the inhibition of the isoprenoid metabolic pathway. Norman et al. (1990) had demonstrated that the site of action would have to be between mevalonate and geranylgeranyl pyrophosphate. This would establish one of the interposed five enzymes, one of which is phosphomevalonate kinase, as the site of action. Somewhat more recent works by Weimer et al. (1992) and Rodney Croteau (1992) suggest, however, that the site of action of clomazone would be found elsewhere.
Within the context of the present invention, an Arabidopsis thaliana cv. Columbia cDNA has been isolated with homology to phosphomevalonate kinase, hereinbelow abbreviated to PMVK, from Saccharomyces cerevisiae (FIG. 1). It was possible to induce this gene in Arabidopsis thaliana cv. Columbia by treatment with the herbicide chlorsulfuron (10 g/ha).
The homology between the Saccharomyces cerevisiae PMVK (=ERG8) and the cDNA isolated from A. thaliana amounts to 44% similarity or 35% identity (see FIG. 1, Bestfit with Wisconsin Package Version 10.1). (ERG8 is the name of the gene encoding phosphomevalonate kinase in yeast (S cerevisiae)). This corresponds for example to the homology between the Saccharomyces cerevisiae mevalonate kinase and the Arabidopsis thaliana mevalonate kinase with a similarity of 45% and an identity of 35%. The function was detected for the Arabidopsis thaliana mevalonate kinase by complementation of the corresponding mutant from Saccharomyces cerevisiae. Moreover, the cDNA isolated within the context of the present invention shows 69% identity with a partial PMVK sequence from Pinus radiate in accordance with SEQ ID NO:5, which is of interest for modifying the isoprenoid content, isoprenoid composition and isoprenoid metabolism of plants (WO 00/36 081). Further partial cDNAs from plants (Medicago trunculata, Accession Number AA660847, see SEQ ID NO:3 and Gossypium hirsutum, Accession Number Al727861, see SEQ ID NO:4) have been isolated as putative PMVKs. Various Arabidopsis spp. sequences (ESTs and genomic sequences) which correspond to the PMVK sequence isolated herein or to parts thereof can be found in databases from various sequencing projects, however, no information is given on the function or importance of these sequences or sequence fragments.
For the first time, the present invention now provides the complete cDNA sequence of a plant phosphomevalonate kinase and describes its use, or the use of the polypeptides encoded thereby, for identifying new herbicidal active compounds.
Subject-matter of the present invention are therefore nucleic acids which encode complete plant phosphomevalonate kinases, with the exception of the partial nucleic acid sequences from Medicago trunculata in accordance with SEQ ID NO:3, Gossypium hirsutum in accordance with SEQ ID NO:4 and Pinus radiata in accordance with SEQ ID NO:5.
Subject-matter of the present invention are, in particular, nucleic acids which encode the Arabidopsis thaliana phosphomevalonate kinase.
Subject-matter of the present invention are very particularly nucleic acids which encode the Arabidopsis thaliana phosphomevalonate kinase and are described under SEQ ID NO:1 and/or encode a polypeptide in accordance with SEQ ID NO:2 or fragments thereof.